Discoidin I from Dictyostelium discoideum and Interactions with oligosaccharides: specificity, affinity, crystal structures, and comparison with discoidin II.

Abstract

Discoidin I (DiscI) and discoidin II (DiscII) are N-acetylgalactosamine (GalNAc)-binding proteins from Dictyostelium discoideum. They consist of two domains: an N-terminal discoidin domain and a C-terminal H-type lectin domain. They were cloned and expressed in high yield in recombinant form in Escherichia coli. Although both lectins bind galactose (Gal) and GalNAc, glycan array experiments performed on the recombinant proteins displayed strong differences in their specificity for oligosaccharides. DiscI and DiscII bind preferentially to Gal/GalNAcbeta1-3Gal/GalNAc-containing and Gal/GalNAcbeta1-4GlcNAcbeta1-6Gal/GalNAc-containing glycans, respectively. The affinity of the interaction of DiscI with monosaccharides and disaccharides was evaluated using isothermal titration calorimetry experiments. The three-dimensional structures of native DiscI and its complexes with GalNAc, GalNAcbeta1-3Gal, and Galbeta1-3GalNAc were solved by X-ray crystallography. DiscI forms trimers with involvement of calcium at the monomer interface. The N-terminal discoidin domain presents a structural similarity to F-type lectins such as the eel agglutinin, where an amphiphilic binding pocket suggests possible carbohydrate-binding activity. In the C-terminal H-type lectin domain, the GalNAc residue establishes specific hydrogen bonds that explain the observed affinity (K(d)=3x10(-4) M). The different specificities of DiscI and DiscII for oligosaccharides were rationalized from the different structures obtained by either X-ray crystallography or molecular modeling.

A: DiscI at 0.04 μg ml-1 and B: DiscII at 0.5 μgml-1 as measured by fluorescence intensity. Complete results including comprehensive oligosaccharide list (Plate Array) are available from the Consortium of Functional Glycomics (http://www.functionalglycomics.org/).

A: Results for GalNAc monosaccharide (10 mM) binding to DiscI. B: Results for Galβ1,3GalNAc disaccharide (2 mM) binding to DiscI. Experiments with 0.15 mM of protein in 20 mM Tris/HCl pH 7.5 and 150 mM NaCl buffer solution at 25°C. Top: data from 30 automatic injections of sugar of 10 μl each into the cell containing DiscI. Bottom: plot of the total heat released as a function of ligand concentration for the titration shown above (squares). The solid line represents the best least-square fit for the obtained data.

A: Crystal structure of the DiscI trimer coloured by chain with the N-terminal domain in light colours and C-terminal domain in dark colours. MPD and GalNAc ligands are represented in ball and sticks. Calcium ions are represented as spheres according to chain colours and nickel ions as a yellow sphere. B: Schematic representation of DiscI topology.

A: Blow up of the glycerol/MPD binding site of DiscI (green) and DiscII (cyan) respectively. B: Blow up of fucose binding site of AAA (magenta) and the F-Lectin from striped Bass (salmon) in the same orientation than discoidins.

A: Representation of the maximum-likelihood weighted 2mFo-DFc electron density around the GalNAc complexed to DiscI contoured at 1σ (0.41 eA3). Hydrogen bonds are represented as dotted lines and waters as magenta spheres. B: Overlay of the GalNAc binding site of DiscI (cyan/marine), DiscII (green/yellow) and HPA (salmon/magenta). The residues are labelled according to their polypeptide chain and number.

Representation of the maximum-likelihood weighted 2mFo-DFc electron density around the GalNAcβ1-3Gal (A) and Galβ1-3GalNAc (B) complexed to DiscI contoured at 1σ (0.44 and 0.45 eA3 respectively). Hydrogen bonds are represented as dotted lines and waters as magenta spheres. C: Overlay of the interactions of the disaccharides with DiscI. The Galβ1-3GalNAc and GalNAcβ1-3Gal disaccharides are coloured in light and dark green respectively. The residues are labelled according to their polypeptide chain and number.

A: Cartoon representation of the superimposition of DiscI (light green/green), DiscII (cyan/blue) and HPA (salmon/magenta) crystal structures. Zoom on one binding site form by two monomers coloured by chain and representation of the GalNAc moiety bound to DiscI in ball and sticks. Surface representation of the binding site of DiscI (B), DiscII (C) and HPA (D). The disaccharides, the modelled trisaccharide and Forsman antigen bound to DiscI, DiscII and HPA (PDB 2CGY) respectively are represented in ball and stick. The Galβ1-3GalNAc and GalNAcβ1-3Gal disaccharides are coloured in light and dark green respectively.